1. Field of the Invention
This invention generally relates to methods and systems for distinguishing between materials having similar spectra. Certain embodiments relate to a computer-implemented method that includes determining which material(s) are associated with a ratio between output signals generated by detecting spectra for a single event in two or more detection windows.
2. Description of the Related Art
Spectroscopic techniques are widely employed in the analysis of chemical and biological systems. Most often, these techniques involve the absorption or emission of electromagnetic radiation by the material of interest. In many cases, scanning the entire relevant section of the spectrum being studied is performed at a slow rate to provide the most accurate measure of absorption or emission. In other systems, however, it is only necessary to examine a specific portion of the spectrum in order to qualify or quantify the parameter under consideration. This examination may be used if, for example, the number of samples is relatively large or the samples must be analyzed relatively quickly. In such situations, the use of small spectral “snapshots” may increase sample throughput by decreasing the amount of raw data that is processed and analyzed.
One such application is in the field of microarrays, which is a technology exploited by a large number of disciplines including the combinatorial chemistry and biological assay industries. One company, Luminex Corp. of Austin, Tex., has developed a system in which biological assays are performed on the surface of variously colored fluorescent microspheres. One example of such a system is illustrated in U.S. Pat. No. 5,981,180 to Chandler et al., which is incorporated by reference as if fully set forth herein. These microspheres are interrogated in a fluid flow device by laser excitation and fluorescence detection of each individual microsphere as they pass at relatively high speed through a detection zone. Such a system is capable of analyzing thousands of microspheres a second, with each microsphere emitting several distinct, detectable signals. Obviously, taking a complete spectrum and interpreting and decoding the signals from each of the many thousands or millions of microspheres would generate an unmanageable quantity of data. The system described by Chandler et al., however, accomplishes management of the data by detecting only the fluorescence in particular “windows,” which are relatively short (e.g., about 20 nm to about 40 nm), continuous portions of the overall spectral emission from the microsphere. As such, rather than generating a complete fluorescence spectrum for each microsphere, this system generates only a single value (which correlates to the intensity of the signal) for each window. These values may be easily exported to a database for further analysis.
In the above-mentioned system, fluorescent dyes are absorbed into the microspheres and/or bound to the surface of the microspheres. The dyes are chosen based on their ability to emit light in the wavelength of the chosen window. Further, the windows are spaced, and the dyes are designed to minimize, and preferably to eliminate, the overlap of a dye's fluorescent signal within adjacent windows. By employing two windows and two dyes, each at 10 different concentrations, there would thus be 100 fluorescently distinguishable microsphere sets.
Another example of an assay method is illustrated in U.S. Pat. No. 4,717,655 to Fulwyler, which is incorporated by reference as if fully set forth herein. In particular, Fulwyler describes a method of distinguishing multiple subpopulations of cells that includes labeling particles with two or more marking agents. These particles are marked in a plurality of different pre-selected ratios of the agents ranging between zero percent and one hundred percent of each agent. Each such agent has distinguishing, quantifiable marking characteristics. In other words, each fluorochrome has distinct emission and/or excitation spectra in specifically designed color bands. The differently labeled particles are mixed with cells suspected of having specific receptors for the differently labeled particles. Each cell is analyzed to determine the ratio of any two identifiable marking characteristics associated with each cell so that it can be classified in a subpopulation category if its ratio of marking characteristics is related to one of the pre-selected ratios of marking agents. Therefore, the method utilizes ratios to distinguish differently labeled particles by detecting a signal from each of two dyes.
In either system or method described above, there are several ways in which the number of distinguishable sets can be expanded. The use of a different sized microsphere, which can be distinguished on the basis of light scatter, will effectively double the number of sets. Another is to increase the number of distinguishable intensities for each dye. For example, if 15 different dye intensities were possible rather than 10 in the example case, then 225 sets would be achievable. A third method would be to add a third window, and subsequently a third dye, or even more, which would exponentially increase the number of sets. Each of these methods has been successfully tested and are being used to varying degrees. However, each adds a layer of complexity to the system, which can greatly add to the expense or difficulty of producing the platform.